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PubMed2001

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol.

Infants with the cholesterol synthesis defect Smith- Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3 beta,7 alpha-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease.--Björkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J. Lipid Res. 2001. 42: 366--371.

Adolescent↗
PubMed1984

Regulation of cholesterol biosynthesis and esterification by 25-hydroxycholesterol in a macrophage-like cell line: uncoupling by progesterone.

The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-like cell line P388D1. Since 25-hydroxycholesterol rapidly stimulated incorporation of [3H]oleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMG-CoA reductase. The experimental results show that progesterone, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), when added together with 25-hydroxycholesterol, abolished the increased cholesterol esterification without affecting the inhibition of HMG-CoA reductase by 25-hydroxycholesterol. Thus, uncoupling cholesterol esterification had no effect on 25-hydroxycholesterol's ability to inhibit HMG-CoA reductase. Unexpectedly, pretreatment of P388D1 cells with 25-hydroxycholesterol resulted in no elevation of ACAT activity as measured in broken cell preparations. Therefore, the possibility that 25-hydroxycholesterol stimulated cholesteryl ester formation by increasing the amount of cholesterol available for esterification, rather than by acting directly on ACAT activity, was considered. Labeling experiments using [14C]-cholesterol have provided evidence for this assumption.

Acetates↗
PubMed1995

25-Hydroxycholesterol stimulates sphingomyelin synthesis in Chinese hamster ovary cells.

In several experimental and pathological conditions the cellular concentrations of cholesterol and sphingomyelin (SM) change coordinately. In an effort to identify factors mediating co-regulation, a class of suppressors of cholesterol synthesis, generically termed oxysterols, were tested for effects on sphingolipid synthesis in Chinese hamster ovary (CHO) cells. 25-Hydroxycholesterol was found to stimulate [3H]serine, [1-3H]palmitate, and [methyl-3H]choline incorporation into sphingomyelin 2- to 3-fold and increase sphingomyelin mass significantly, but did not influence synthesis of other major phospholipids. Maximal labeling of sphingomyelin by [3H]serine was observed 4-6 h after oxysterol addition, and coincided with inhibition of transcription of sterol-regulated genes and activation of cholesteryl ester synthesis. 25-Hydroxycholesterol dose-response curves for activation of sphingomyelin synthesis, suppression of sterol-regulated transcription, and activation of cholesteryl ester synthesis were also similar. Stimulation of SM and glucosylceramide synthesis was observed only with 25-hydroxycholesterol; other oxysterols and cholesterol were ineffective or inhibitory. The effects of 25-hydroxycholesterol on sphingolipid synthesis could not be reproduced by low density lipoprotein (LDL), whole serum, or non-lipoprotein cholesterol in the medium, and stimulation by 25-hydroxycholesterol was evident irrespective of a cholesterol source in the medium. 25-Hydroxycholesterol-treated CHO cells displayed enhanced conversion of [3H]sphinganine-labeled ceramide into sphingomyelin. Sphingomyelin synthesis from N-hexanoyl [3-3H]ceramide and N-hexanoyl [3-3H]dihydroceramide was also increased significantly. Consistent with enhanced ceramide conversion to sphingomyelin, ceramide mass was reduced by 20-40% in 25-hydroxycholesterol-treatment. However, in vitro activity of sphingomyelin synthase (assayed with short-chain ceramide) was not increased in membranes from oxysterol-treated cells. Stimulation of sphingolipid synthesis by 25-hydroxycholesterol is temporally related to effects of this oxysterol on cholesterol metabolism, and is the result of enhanced conversion of ceramide to SM.

Animals↗
PubMed1994

Alterations of Ca++ uptake and lipid content in cultured human arterial smooth muscle cells treated with 26-hydroxycholesterol.

The effects of 26-hydroxycholesterol on 45Ca++ uptake, [14C]cholesterol uptake, [14C] acetate incorporation and the concentrations of 26-hydroxycholesterol and cholesterol in the plasma membrane were studied in cultured human smooth muscle cells isolated from umbilical arteries. The results showed that: (1) 26-hydroxycholesterol enhanced markedly 45Ca++ uptake and the enhancement was not diminished by nifedipine; (2) 26-hydroxycholesterol decreased cholesterol content in plasma membrane by inhibiting cholesterol uptake and synthesis, and [14C]cholesterol uptake was not LDL-receptor dependent; (3) 26-hydroxycholesterol induced a shift of [14C] acetate from cholesterol into phospholipid synthesis, but the radioactive incorporation into triglyceride and cholesterol ester was inhibited by 26-hydroxycholesterol; and (4) 26-hydroxycholesterol inserted itself into the plasma membrane. We suggested that 26-hydroxycholesterol changes the composition of membrane lipids with a consequential alteration of membrane permeability to Ca++.

Biological Transport↗
PubMed2002

Reduction of plasma 24S-hydroxycholesterol (cerebrosterol) levels using high-dosage simvastatin in patients with hypercholesterolemia: evidence that simvastatin affects cholesterol metabolism in the human brain.

BACKGROUND: Previous studies have shown that patients with early onset of Alzheimer disease and vascular dementia have higher levels of circulating brain-derived 24S-hydroxycholesterol (cerebrosterol). Two recent epidemiological studies indicated that treatment with inhibitors of cholesterol synthesis (statins) reduces the incidence of Alzheimer disease. OBJECTIVE: To test the hypothesis that treatment with high-dosage simvastatin reduces circulating levels of 24S-hydroxycholesterol. DESIGN: Prospective, 24-week treatment trial for lowering of cholesterol levels. We conducted assessments at baseline, week 6, and week 24. SETTING: An academic outpatient clinical study. PATIENTS: Eighteen patients who met the criteria for hypercholesterolemia. INTERVENTION: Treatment with 80 mg/d of simvastatin at night. MAIN OUTCOME MEASURES: Plasma lipoprotein levels were measured enzymatically; lathosterol, by means of gas chromatography; and 24S-hydroxycholesterol, by means of gas chromatography-mass spectrometry. RESULTS: Simvastatin reduced total plasma cholesterol levels by 36% and 35% after 6 and 24 weeks, respectively (P<.001). Lathosterol levels were reduced by 74% and 72%, respectively, and the ratio of lathosterol to cholesterol, an indicator of whole-body cholesterol synthesis, was reduced by 60% and 61%, respectively (P<.001). Plasma 24S-hydroxycholesterol levels were lowered by 45% and 53%, respectively (P<.001). The ratio of 24S-hydroxycholesterol to cholesterol also decreased significantly (-12% [P=.01] and -23% [P<.002], respectively). The further reduction of 24S-hydroxycholesterol levels and its ratio to cholesterol from weeks 6 to 24 was also significant (P=.02 for both). CONCLUSIONS: The greater reduction of plasma concentrations of 24S-hydroxycholesterol compared with cholesterol indicates that simvastatin in a dosage of 80 mg/d reduces cholesterol turnover in the brain. The present results might describe a possible mechanism of how long-term treatment with statins could reduce the incidence of Alzheimer disease.

Administration, Oral↗
PubMed2002

Cholesterol, hydroxycholesterols, and bile acids.

Although a variety of oxidation products of cholesterol occur in vitro, enzyme-catalyzed oxidations can occur at only 5 sites on the cholesterol molecule: C7alpha, C22R, C24S, C25, and C27. The genes coding for the synthesis of these enzymes were cloned, the tissue expressions of the mRNAs were identified, and the enzymes were characterized. The biologic properties of the hydroxycholesterol molecules that are initially generated and their metabolites are under study. Downregulation of cholesterol synthesis via the SREBP/SCAP regulatory pathway is common to the initial hydroxycholesterols, but more variations exist with respect to these intermediates functioning as ligands for the nuclear receptor LXRalpha. Because this receptor regulates the expression of cholesterol 7alpha-hydroxylase and ABC transporter proteins, hydroxycholesterols and their intermediate steroid metabolites modulate a number of biologic processes. Metabolism of 22S-hydroxycholesterol to steroid hormones differs from that of the other hydroxycholesterols which form mostly steroid acidic products, otherwise known as bile acids. In vivo estimates of their production rates in intact humans indicate that 24S and 25-hydroxycholesterol account for no more than 7% of total bile acid production per day. Current evidence indicates that cholesterol 7alpha-hydroxycholesterol generated in the liver is the major source of bile acids in older adults. It is also known that the cholesterol 27-hydroxylation pathway is the only one expressed in fetal and neonatal life. Precisely when the proportions contributed by these two metabolic pathways to bile acid synthesis begin to shift and the role of the cholesterol 27-hydroxylase pathway in reverse cholesterol transport mandate further study.

Animals↗
PubMed1994

Effect of biliary drainage on serum 7 alpha-hydroxycholesterol level in patients with obstructive jaundice.

Percutaneous transhepatic biliary drainage was performed in 15 patients with obstructive jaundice and the serum 7 alpha-hydroxycholesterol level was monitored for 2 weeks by capillary gas-liquid chromatography-mass spectrometry. Before biliary drainage, the serum 7 alpha-hydroxycholesterol level was 130 +/- 52 pmole/ml, which was significantly lower than the normal value (238 +/- 100 pmole/ml). After biliary drainage, the serum 7 alpha-hydroxycholesterol level was decreased to 117 +/- 48 pmole/ml on the first day. There were two groups of patients as classified by the behavior of the serum 7 alpha-hydroxycholesterol level after the third day. In one group of patients (responders), the serum level increased significantly up to 525 +/- 83 pmole/ml at the end of the 2 weeks. In another group (nonresponders), the serum level did not increase and remained 145 +/- 86 pmole/ml. The serum bilirubin level tended to improve faster in the responders (decline index = -0.234 +/- 0.152) than in the nonresponders (decline index = -0.057 +/- 0.067; P < 0.05) and a statistically significant correlation was found between the decline index and the total 7 alpha-hydroxycholesterol level on Days 7 or 14. As the serum 7 alpha-hydroxycholesterol level is considered to reflect hepatic bile acid synthesis, which is one of the important functions of the liver, we conclude that the increased serum 7 alpha-hydroxycholesterol level after biliary drainage in the responders indicates recovered liver function and that the low serum level in the nonresponders suggests more severe hepatocellular damage in patients with obstructive jaundice.

Aged↗
PubMed1992

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis in Syrian hamster C100 cells by mevinolin, 25-hydroxycholesterol, and mevalonate: the role of posttranscriptional control.

C100 is a baby hamster kidney cell line that expresses high levels of HMG-CoA reductase relative to its parental cell line SV28. In this study the effects of the oxysterol 25-hydroxycholesterol and mevalonate on the mRNA level and rate of synthesis for HMG-CoA reductase were evaluated in C100 cells treated with mevinolin, a competitive inhibitor of HMG-CoA reductase. The addition of 25-hydroxycholesterol to the cell culture medium resulted in a fourfold decrease in both the rate of synthesis and mRNA level for HMG-CoA reductase. Mevalonate at a concentration of 0.4 mM, when added to mevinolin-treated C100 cells, produced no apparent reduction in HMG-CoA reductase mRNA levels and only a small (25%) decline in HMG-CoA synthesis. Mevalonate (0.4 mM) added to 25-hydroxycholesterol-treated cells resulted in no further reduction in the HMG-CoA reductase mRNA level when compared to cells treated with 25-hydroxycholesterol alone, but produced an additional 30-fold decrease in the rate of HMG-CoA reductase synthesis. Degradation of HMG-CoA reductase was rapid in the presence (t1/2 = 1.34 h) or absence (t1/2 = 1.17 h) of mevinolin and was not changed significantly by adding either 25-hydroxycholesterol, alone (t1/2 = 1.30 h) or both 25-hydroxycholesterol and mevalonate (t1/2 = 1.30 h) to mevinolin-treated cells. This study demonstrates that mevalonate and 25-hydroxycholesterol act synergistically in the presence of mevinolin to achieve a greater degree of suppression in the rate of HMG-CoA reductase synthesis than can be accounted for by their individual effects on HMG-CoA reductase mRNA. In addition, the data suggest that mevalonate affects the synthesis of HMG-CoA reductase at a yet unidentified posttranscriptional control site.

Amino Acid Sequence↗
PubMed1983

Prevention of the hyperlipidemic serum or LDL-induced cellular cholesterol ester accumulation by 22-hydroxycholesterol and its analogue.

Incubation of monkey arterial smooth muscle cells with hyperlipidemic serum or low-density lipoproteins (LDL) produces intracellular cholesterol ester accumulation. Increased esterification of free cholesterol within the cell may account for this effect. To examine such a possibility, oxygenated sterols were used to block cholesterol esterification. The increased esterification of free cholesterol by smooth muscle cells and human skin fibroblasts, through their exposure to hyperlipidemic lipoproteins, was inhibited by 22-hydroxycholesterol and its analogue, SC-32561 (22-hydroxy-25-fluorocholesterol). The hyperlipidemic LDL-stimulated elevation in the cholesterol ester content of the smooth muscle cells was also prevented by these sterols. This reduction in cellular cholesterol ester did not coincide with an increase in free cholesterol. 22-Hydroxycholesterol also blocked the stimulation of the esterification of cholesterol due to 25-hydroxycholesterol. In the absence of lipoproteins, 22-hydroxycholesterol and SC-32561 had a minor effect on the incorporation of [14C]oleate into cholesterol esters, and efficiently reduced sterol synthesis in fibroblasts and smooth muscle cells. 22-Hydroxycholesterol and SC-32561 had the additional effect of lowering the number of cell-surface LDL receptors to a greater extent than did hyperlipidemic LDL. The presence of 22-hydroxycholesterol did not alter the interaction of normal LDL with the receptor. Oxygenated sterols recovered in the cell represented 4-21% of the total sterol content. The level of intracellular oxygenated sterols was significantly reduced by the presence of lipoproteins in the culture media. Due to the multiple effects of the oxysterols, they were not effective as tools in determining the contribution of acyl-coenzyme A:cholesterol acyltransferase enzyme activity to the intracellular pool of cholesterol esters. These results indicated that 22-hydroxycholesterol and SC-32561 effectively blocked the hyperlipidemic LDL-stimulated increase in smooth muscle cell cholesterol ester content by lowering cholesterol esterification, reducing cholesterol synthesis and down-regulating LDL cell-surface receptors.

Animals↗
PubMed1987

Determination of some hydroxycholesterols in human serum samples.

The simultaneous determination of some hydroxycholesterols in human serum samples is described. The procedure is based on hydrolysis and extraction of these compounds in serum samples, followed by removal of especially cholesterol (making use of reversed-phase high-performance liquid chromatography) and derivatization of the purified compounds to their trimethylsilyl ethers and subsequent gas chromatography using flame ionization detection. Serum levels of 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol and 26-hydroxycholesterol were determined in several groups of patients: normals, untreated patients suffering from cerebrotendinous xanthomatosis, patients suffering from cerebrotendinous xanthomatosis and treated with either chenodeoxycholic acid or cholic acid in an effective dose, patients suffering from cerebro-hepato-renal syndrome, patients suffering from hypercholesterolemia and treated with cholestyramine for prolonged periods and one patient presumed to be suffering from an inborn error of metabolism in bile acid synthesis. It can be concluded that the 7 alpha-hydroxycholesterol concentration in serum is a good parameter for establishing disorders involving the metabolic conversion of 7 alpha-hydroxycholesterol towards bile acids. In addition, 26-hydroxycholesterol levels in patients suffering from cerebrotendinous xanthomatosis are beyond detectable limits, even during treatment with bile acids in an effective dose, whereas in all other conditions this compound is substantially present.

Chromatography, Gas↗
PubMed2006

Lipoprotein distribution and biological variation of 24S- and 27-hydroxycholesterol in healthy volunteers.

24S- and 27-hydroxycholesterol are obligatory intermediates of cholesterol catabolism and play an important role in the maintenance of whole-body cholesterol homeostasis. Using an HPLC-MS method for oxysterol quantification, the distribution of esterified and unesterified oxysterols in lipoprotein subfractions as well as the influence of daytime, food intake and menstrual cycle on oxysterol concentrations were investigated in healthy volunteers. Moreover, reference intervals for 24S- and 27-hydroxycholesterol in plasma as well as the corresponding levels for 27-hydroxycholesterol in the HDL subfraction were established in 100 healthy volunteers. Both circulating oxysterols are mainly transported in association with HDL and LDL--primarily in the esterified form. No significant diurnal changes and no variations during menstrual cycle of either absolute or cholesterol-related plasma levels were detected. In contrast to 24S-hydroxycholesterol in plasma and 27-hydroxycholesterol in the HDL subfraction, the 95% reference intervals of 27-hydroxycholesterol both in plasma and the non-HDL subfraction were higher in males than in females. The concentrations of 27-hydroxycholesterol in plasma and the non-HDL subfraction showed strong positive correlations with the concentrations of cholesterol, non-HDL cholesterol and triglycerides. Our data on the lipoprotein distribution of oxysterols as well as on their intra- and inter-individual variation set the stage for future clinical studies.

Adult↗
PubMed2004

Does brain specific 24S-hydroxycholesterol in plasma indicate the disruption of the blood-brain barrier in patients with ischemic stroke?

Determination of plasma 24S-hydroxycholesterol, which is produced almost exclusively in the brain and is released only in small amounts into circulation under physiological conditions, might be a marker to monitor non-invasively the time course and the extent of the disintegration of the blood-brain barrier after cerebral ischemia. We investigated the plasma concentrations for 4 to 10 days of 24S-hydroxycholesterol, and compared the concentration with the liver-specific oxysterol 7alpha-hydroxycholesterol, the ubiquitously produced 27-hydroxycholesterol, and cholesterol itself in six patients who were admitted to the hospital within 24 h after symptoms of stroke. Quantification of oxysterols was performed by isotope dilution mass spectrometry and cholesterol by gas-liquid chromatography. Initial concentrations of cholesterol, 24S-, 7alpha-, and 27-hydroxycholesterol in stroke patients were not different from data of healthy controls given in the literature. During the following days, no changes could be observed in the concentrations of cholesterol nor in the other oxysterols. Particularly the brain specific 24S-hydroxycholesterol was very constant and showed only minimal changes. Furthermore, comparison of patients with extended or small lesions did not reveal any differences in the concentrations of oxysterols. Therefore, circulating levels of 24S-hydroxycholesterol are supposed to be only of limited value for monitoring the brain-blood barrier function in patients with acute ischemic stroke.

Adult↗
PubMed1998

Biochemical characterization of the 7alpha-hydroxylase activities towards 27-hydroxycholesterol and dehydroepiandrosterone in pig liver microsomes.

Microsomal cytochrome P-450 catalyzing the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone was partially purified from pig liver. This enzyme fraction also catalyzed 7alpha-hydroxylation of 25-hydroxycholesterol and pregnenolone but did not 7alpha-hydroxylate cholesterol or testosterone. Studies with extrahepatic tissues have suggested the possibility of one common enzyme responsible for the 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone. A series of experiments was performed to study if there are one or several enzymes 7alpha-hydroxylating these steroids in the liver. The activities towards the two substrates copurified but the ratio between 27-hydroxycholesterol and dehydroepiandrosterone 7alpha-hydroxylation varied considerably in different purification steps and between different preparations. The enzyme inhibitors disulfiram, N-bromosuccinimide, ketoconazole, metyrapone and alpha-naphthoflavone affected the activities in a similar way. Dehydroepiandrosterone inhibited 27-hydroxycholesterol 7alpha-hydroxylation whereas 27-hydroxycholesterol had almost no inhibitory effect on dehydroepiandrosterone 7alpha-hydroxylation. Experiments to examine the nature of inhibition by dehydroepiandrosterone indicated that the two steroids did not compete for the same active site. The results of this study do not rule out the possibility of one single enzyme catalyzing 7alpha-hydroxylation of the two steroids. However, taken together the data suggest that hepatic microsomal 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two, probably closely related, enzymes. (c) 1998 Elsevier Science B. V.

Animals↗
PubMed2000

Formation of oxysterols from different pools of cholesterol as studied by stable isotope technique: cerebral origin of most circulating 24S-hydroxycholesterol in rats, but not in mice.

In order to study the origin of different oxysterols in the circulation, in particular 24S-hydroxycholesterol, different pools of cholesterol in rat and mouse were labelled by feeding the animals with a diet supplemented with 0.3 or 0.5% hexadeuterium-labelled cholesterol, respectively, for 10 days. The incorporation of deuterium label in cholesterol and different oxysterols was measured by combined gas chromatography-mass spectrometry in selected tissues and in the circulation. In both rat and mouse, a high incorporation of label was found in cholesterol present in serum and liver (up to 77%). Incorporation of label was similar in 7 alpha- and 7 beta-hydroxycholesterol of the same origin. There was no significant incorporation of deuterium in brain cholesterol, and little or no incorporation in the brain oxysterols investigated, in both animals. In the testis, the incorporation of the deuterium label in cholesterol was less than half of that in the liver, with similarly reduced labelling of the testicular oxysterols. 24S-Hydroxycholesterol in the circulation contained a deuterium content that was about 50% of that of serum and liver cholesterol in the mouse experiment and about 30% in the rat experiment. Thus, about 50% of circulating 24S-hydroxycholesterol in the mouse and about 70% of this fraction in the rat must originate from pools of cholesterol that are not in equilibrium with plasma and liver cholesterol. The liver is probably responsible for a considerable part of the extracerebral formation of 24S-hydroxycholesterol, since this organ contained detectable amounts of 24S-hydroxycholesterol with a relatively high incorporation of deuterium in both animal species. The results are consistent with a cerebral origin of more than half of the 24S-hydroxycholesterol in the circulation of rats, but not in mice.

Adrenal Glands↗
PubMed1999

Alpha-tocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells.

The cytotoxicity of oxysterols including 7-ketocholesterol, alpha-epoxide, cholestanetriol and 25-hydroxycholesterol and the possible protecting effect of alpha-tocopherol on cholestanetriol and 25-hydroxycholesterol-induced cytotoxicity were investigated in primary cultures of porcine ovarian granulosa cells. Cell viability as determined by % trypan blue staining and mitochondrial function as determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction were decreased significantly after 24 h exposure to 2.5 microM alpha-epoxide, cholestanetriol and 25-hydroxycholesterol. 7-Ketocholesterol (2.5 microM) did not affect cell viability or mitochondrial function under the same culture conditions. The specific activities of catalase and superoxide dismutase, two antioxidant defense enzymes were increased significantly (p < 0.01) following 24 h exposure to 2.5 microM concentrations of cholestanetriol while only superoxide dismutase was increased in 25-hydroxycholesterol-treated cells (p < 0.001). Specific activity of glutathione peroxidase was unchanged relative to control cells. Levels of thiobarbituric acid reactive substances remained unchanged after exposure to 7-ketocholesterol, alpha-epoxide, cholestanetriol, 25-hydroxycholesterol and cholesterol. Administration of 1 microM alpha-tocopherol to the culture medium significantly improved cell viability and restored both superoxide dismutase and catalase activities to control levels in cholestanetriol -treated cells and only superoxide dismutase in 25-hydroxycholesterol-treated cells. These studies suggest that the cytotoxic nature of physiologically relevant concentrations of cholestanetriol and 25-hydroxycholesterol in granulosa cells is in part due to oxidative stress, but it may be reduced in the presence of alpha-tocopherol.

Animals↗
PubMed2001

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Cholesterol and bile acid synthesis in Hep G2 cells. Metabolic effects of 26- and 7 alpha-hydroxycholesterol.

1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed.

Bile Acids and Salts↗
PubMed2003

24S-hydroxycholesterol: a marker of brain cholesterol metabolism.

The enzymatic conversion of CNS cholesterol to 24S-hydroxycholesterol, which readily crosses the blood-brain barrier, is the major pathway for brain cholesterol elimination and brain cholesterol homeostasis maintenance. The enzyme mediating this conversion has been characterized at the molecular level (CYP46) and is mainly located in neurons. Like other oxysterols, 24S-hydroxycholesterol is efficiently converted into normal bile acids or excreted in bile in its sulfated and glucuronidated form. Levels of 24S-hydroxycholesterol in the circulation decrease with age in infants and children. In adults, however, the levels appear to be stable. There is accumulating evidence pointing toward a potentially important link between cholesterol, beta-amyloid, and Alzheimer's disease. Concentrations of 24S-hydroxycholesterol in plasma and cerebrospinal fluid (CSF) are significantly higher in Alzheimer's disease and vascular demented patients at early stages of the disease compared to healthy subjects. Variations in genetic background, time of disease onset, and severity of dementia are potential sources of variance. Inhibitors of cholesterol biosynthesis, also termed statins, seem to have a reductive influence on the generation of the amyloid precursor protein, the neuronal secretion of beta-amyloid, and on de novo cholesterol synthesis. Recent epidemiological studies indicate that the prevalence of diagnosed AD and vascular dementia is reduced among people taking statins for a longer period of time. High-dose simvastatin treatment (80 mg/day) in patients with hypercholesterolemia leads to a significant decrease in brain-specific serum 24S-hydroxycholesterol concentrations and indicates a diminished cholesterol metabolism in the brain. CSF levels of cholesterol and lathosterol, a cholesterol precursor considered to be an indicator for cholesterol neogenesis, were significantly decreased in statin-treated subjects compared to non-treated normo- and hypercholesterolemic subjects. Also, CSF concentrations of 24S-hydroxycholesterol were significantly lower in statin-treated patients compared to normocholesterolemic subjects. Treatment with high-dose simvastatin in normocholesterolemic Alzheimer patients for 26 weeks at early stages of the disease results in a significant decrease in Abeta-levels in cerebrospinal fluid. This decrease correlates with the reduction of 24S-hydroxycholesterol.

Adolescent↗